The ubiquitous bacterial second messenger cyclic diguanylate (c-di-GMP) coordinates diverse cellular processes through its downstream receptors. However, whether c-di-GMP participates in regulating nitrate assimilation is unclear. Here, we found that NasT, an antiterminator involved in nitrate assimilation in Pseudomonas putida, specifically bound c-di-GMP. NasT was essential for expressing the nirBD operon encoding nitrite reductase during nitrate assimilation. High-level c-di-GMP inhibited the binding of NasT to the leading RNA of nirBD operon (NalA), thus attenuating the antitermination function of NasT, resulting in decreased nirBD expression and nitrite reductase activity, which in turn led to increased nitrite accumulation in cells and its export. Molecular docking and point mutation assays revealed five residues in NasT (R70, Q72, D123, K127 and R140) involved in c-di-GMP-binding, of which R140 was essential for both c-di-GMP-binding and NalA-binding. Three diguanylate cyclases (c-di-GMP synthetases) were found to interact with NasT and inhibited nirBD expression, including WspR, PP_2557, and PP_4405. Besides, the c-di-GMP-binding ability of NasT was conserved in the other three representative Pseudomonas species, including P. aeruginosa, P. fluorescens and P. syringae. Our findings provide new insights into nitrate assimilation regulation by revealing the mechanism by which c-di-GMP inhibits nitrate assimilation via NasT.